Effects of pharmacological inhibition of the sodium‐dependent phosphate cotransporter 2b (NPT2b) on intestinal phosphate absorption in mouse and rat models

Abstract An excess phosphate burden in renal disease has pathological consequences for bone, kidney, and heart. Therapies to decrease intestinal phosphate absorption have been used to address the problem, but with limited success. Here, we describe the in vivo effects of a novel potent inhibitor of the intestinal sodium‐dependent phosphate cotransporter NPT2b, LY3358966. Following treatment with LY3358966, phosphate uptake into plasma 15 min following an oral dose of radiolabeled phosphate was decreased 74% and 22% in mice and rats, respectively, indicating NPT2b plays a much more dominant role in mice than rats. Following the treatment with LY3358966 and radiolabeled phosphate, mouse feces were collected for 48 h to determine the ability of LY3358966 to inhibit phosphate absorption. Compared to vehicle‐treated animals, there was a significant increase in radiolabeled phosphate recovered in feces (8.6% of the dose, p < .0001). Similar studies performed in rats also increased phosphate recovered in feces (5.3% of the dose, p < .05). When used in combination with the phosphate binder sevelamer in rats, there was a further small, but not significant, increase in fecal phosphate. In conclusion, LY3358966 revealed a more prominent role for NPT2b on acute intestinal phosphate uptake into plasma in mice than rats. However, the modest effects on total intestinal phosphate absorption observed in mice and rats with LY3359866 when used alone or in combination with sevelamer highlights the challenge to identify new more effective therapeutic targets and/or drug combinations to treat the phosphate burden in patients with renal disease.


| INTRODUC TI ON
In healthy adults, the amount of dietary phosphate absorbed is balanced by phosphate excreted through the kidney. 1 In chronic kidney disease (CKD) patients, the ability to excrete phosphate through the kidney is compromised and several compensatory mechanisms are activated to help maintain serum phosphate levels within the normal range. 2 These include an elevation in FGF23 and PTH, which decrease the reabsorption of filtered phosphate in the kidneys, and a reduction in 1,25-dihydroxyvitamin D, which decreases intestinal NPT2b expression and phosphate absorption. However, in advanced CKD and end-stage renal disease (ESRD), the compensatory mechanisms are inadequate, and hyperphosphatemia ensues. 3 Although compensatory mechanisms help attenuate the rise in serum phosphate levels, they along with the increased phosphate burden have detrimental effects on bone, kidney, and heart. 2,4 If one was able to adequately decrease dietary phosphate absorption, this pathological sequence of events may be minimized.
To balance intestinal phosphate absorption with the decrease in renal excretion in CKD and ESRD, dietary phosphate restriction and phosphate binder therapies have been used. Given the high levels of phosphate in Western diets and typical poor compliance encountered with restrictive diets, controlling phosphate by dietary phosphate restriction can be challenging. 5,6 In addition, phosphate binders have limited phosphate binding capacity, which limits their efficacy. 7 Based upon decreases in urinary phosphate or increases in fecal phosphate content with treatment, phosphate binders appear to only inhibit phosphate absorption around 20%-40% for a normal diet. [8][9][10][11] This may explain their limited ability to control FGF23 and PTH levels in CKD. 8,[12][13][14] A small study with severe dietary phosphate restriction that achieved a 50% decrease in urinary phosphate produced a profound 70% decrease in PTH in humans, 15 highlighting the potential for a new therapy that robustly decreases dietary phosphate absorption as a monotherapy or in combination therapy.
The inability to control phosphate levels in CKD and ESRD has led to a search for more effective therapies to limit dietary phosphate absorption. In the human intestine, dietary phosphate is absorbed through active transport and passive diffusion. 16 Current therapies used to limit dietary phosphate absorption decrease the free phosphate concentration in the intestinal lumen, suggesting they would decrease absorption through an effect on passive diffusion. Recently, the NHE3 inhibitor, tenapanor, was shown to inhibit intestinal phosphate absorption by blocking the paracellular diffusional pathway. 17,18 The need for more effective therapies has led to a search for inhibitors of active phosphate transport. Based upon preclinical animal models, 19,20 the intestinal high-affinity sodium-dependent phosphate cotransporter 2b (NPT2b) with a K P i m = 10 µM 21 is thought to play a prominent role in intestinal phosphate active transport. At the millimolar phosphate concentrations found in the human intestine after a meal, 16 NPT2b should transport phosphate at its maximal velocity.
More recently, studies in rats and humans have implicated the ubiquitously expressed, low-affinity sodium-dependent phosphate transporters PiT1 and possibly PiT2 in intestinal phosphate absorption. 22,23 The contribution of passive diffusional transport and the various sodium-dependent active transporters in phosphate absorption is uncertain in both preclinical models and humans.
Complicating our understanding, their contributions undoubtedly vary under different conditions, for example, the concentration of free phosphate in the intestine, the age of the animals or individuals, their health status (healthy, CKD or ESRD), the 1,25-dihydroxyvitamin D status (upregulates active transport and NPT2b 24,25 ), and counter-regulation of other pathways in response to therapy. As it is unlikely any one therapy will effectively control the phosphate burden in the more challenging patients, it is also important to understand how different therapies work in combination.
To further address these issues, a medicinal chemistry campaign was used to identify a potent NPT2b inhibitor, LY3358966. The compound was used to explore the role of NPT2b-mediated active transport in phosphate absorption in two preclinical animal models under carefully controlled conditions. In addition, the ability of the inhibitor to work in combination with the phosphate binder, sevelamer, was also investigated.

| LY3358966 formulation
A spray-dried solid dispersion (SDD) containing 30% LY3358966 was made by adding the free base of LY3358966 to poly-1-vinylpyrrolidoneco-vinyl acetate (PVP-VA) in methanol. Two mole equivalents of NaOH were added to the slurry, which was then bath sonicated until a clear yellow solution was formed. The solution was slowly pumped into a spray dryer with a stream of hot nitrogen, resulting in a solid powder that was collected and further dried in a vacuum oven at 50°C. In one study, the solid formulation (LY3358966 SDD) was placed in a capsule, while in other studies, it was dissolved in water before dosing.

| In vivo studies
All animal procedures were approved by and conducted in accordance with the Eli Lilly and Covance Institutional Animal Care and Use Committee guidelines. Mice and rats were fed Teklad Global 14% protein rodent maintenance diet (Envigo) containing 0.6% of phosphorus (0.3% non-phytate phosphorus).

| Gastric emptying assay in mice
Male C57BL/6 mice, approximately 7-8 weeks old (18-21 g), were fasted overnight. Then, the mice were orally administered a single dose of vehicle (water) or varying doses of LY3358966 SDD dissolved in water. Fifteen minutes later, radiolabeled phosphate was given by oral gavage. Another 15 min later, the animals were sacrificed, and intact stomachs were collected and placed in 50-ml conical tubes. Ten milliliter of 1 N NaOH was added to each tube and the stomachs were digested at 37°C overnight. Radioactivity in 100 µl digested stomach homogenate was quantitated by scintillation counting. The percent radiolabeled phosphate recovered in the stomach was calculated as the percentage of dpm recovered in the stomach versus the total dpm of radiolabeled phosphate administered.
At t = 0, the animals were dosed with radiolabeled phosphate as described above and were given free access to food. Feces were collected at 6, 24, and 48 h, digested overnight at 37°C in 1 N NaOH, and the recovered radioactivity was determined. Most of the radiolabeled phosphate was recovered in the first 24 h. In a control study, we demonstrated that 82 ± 1.34% (mean ± SEM, n = 10) of a non-absorbed control, 14 C-polyethylene glycol-4000 (PerkinElmer), could be recovered in the feces by 48 h. In all cases, the investigators collecting feces were blinded to the treatment.

| Inhibition of phosphate absorption by LY3358966 alone or in combination with sevelamer HCl in rats
Male Sprague Dawley rats, 260-287 g for the first study and 274-362 g for the second study, were fasted for 4 h (the first 4 h of the light cycle) then dosed with placebo, a 50 or 150 mg/ kg sevelamer HCl water dispersion, LY3358966 SDD (10 mg/kg API) dissolved in water, or a combination of LY3358966 SDD and sevelamer HCl. Fifteen minutes later, the animals were dosed with radiolabeled phosphate as described above. In the first study, the animals were given free access to food, then feces were collected at 6, 24, and 48 h post radiolabeled phosphate dose. In the second study, the animals were fasted an additional 4 h, then feces, stomach, small intestine, and large intestine were individually collected. The gastrointestinal tract tissues and feces were digested overnight at 37°C in 1 N NaOH, and the recovered radioactivity was determined.

| Statistical analyses
An unpaired Student's t-test with two-tails was utilized for the twogroup comparisons in the capsule study and the mouse fecal study. It was also used for multiple group comparisons in the gastric emptying study to provide a statistical test that optimizes the chance to detect a significant effect. One-way ANOVA with Dunnett's multiple comparisons was utilized for multiple-group comparisons in other studies.

| In vitro inhibition of sodium-dependent cotransporters by LY3358966
LY3358966 (Figure 1) was discovered through a medicinal chemistry campaign. It was tested for its ability to inhibit sodium-dependent 33 P-phosphate uptake in CHO cells over-expressing human NPT2b.
LY3358966 inhibited phosphate uptake in a concentrationdependent manner with an IC 50 of 32.4 nM ( Figure 2; Table 1A).
LY3358966 also inhibited mouse and rat NPT2b expressed in CHO cells with similar potencies (Table 1A).
The in vitro activity of LY3358966 was determined for other members of the human type II sodium phosphate cotransporter family, NPT2a and NPT2c, which are found primarily in the kidney and members of the human type III sodium phosphate cotransporter family, PiT1 and PiT2, which are ubiquitously expressed. LY3358966 was a potent inhibitor of NPT2a and NPT2c ( Figure 2;

| Pharmacokinetics of LY3358966 SDD
A SDD of LY3358966 formulated with PVP-VA (LY3358966 SDD) was dissolved in water and used to determine the pharmacokinetic properties of orally administered LY3358966 in mice. It was administered to mice through an IV route at 1 mg/kg and a P.O.
LY3358966 is highly protein bound. AUC unbound and C max,unbound were estimated to be <2 nM × h and <1 nM, respectively, across all doses.

| Effect of LY3358966 SDD on acute phosphate uptake into plasma in mice and rats
LY3358966 SDD was dissolved in water and then orally gavaged to mice at a dose range from 0.03 to 30 mg/kg API. Following an oral dose of 33 Table 1 0

| Effect of LY3358966 on gastric emptying in mice
To confirm that the decrease in 33 P-phosphate appearing in the plasma of LY3358966-treated animals is due to inhibition of 33 Pacute phosphate uptake, and not retention of 33 P-phosphate in the stomach, LY3358966 SDD was tested for its effect on gastric emptying in mice. LY3358966 at doses up to 100 mg/kg API had no effect on gastric emptying (Figure 4). This is an important finding because numerous compounds in our medicinal chemistry campaign inhibited gastric emptying and thus, were not pursued.

| In vivo effect of LY3358966 SDD on phosphate absorption in mice when dosed as a solid in a capsule
The solubility and formulation of LY335896 are critical for its in vivo activity. A solid dose formulation of LY3358966, which is preferred as a human therapy, was developed. In the case of an NPT2b inhibitor, this necessitates that the solid formulation is solubilized in the stomach, at a pH where LY3358966 has minimal solubility, and remain in solution long enough to inhibit intestinal NPT2b. The ability of a solid SDD of LY3358966 to inhibit intestinal NPT2b was investigated in mice. The solid LY3358966 SDD formulation dosed in a capsule inhibited phosphate absorption (Table 3) comparably to LY3358966 SDD dosed in a solution (p = .46).

| The recovery of radiolabeled phosphate in feces of mice treated with LY3358966 SDD
To explore the ability of LY3358966 to block radiolabeled phosphate absorption longitudinally in mice, three doses of 9 mg/kg API of LY3358966 SDD were administered orally at 4-h intervals. This dose was selected because it is about 8 times higher than the ED 50   The first approach to assess the effect of LY3358966 on intestinal phosphate absorption in rats was to determine 33 P-phosphate excretion in feces collected for 48 h after dosing 33 P-phosphate, analogous to the mouse study. The 48-h fecal recovery of 33 P-phosphate in the placebotreated group was 12.95% of the dose ( Figure 6). The NPT2b inhibitor mediated recovery (difference between placebo and LY3358966-treated groups) was 5.34% (p = .0165). Fifty and 150 mg/kg sevelamer did not increase 33 P-phosphate recovered from the 48-h feces collection (13.43% and 13.10%, respectively, vs. 12.95% in the placebo group, Figure 6).
When 50 or 150 mg/kg sevelamer was combined with LY3358966, there was a very modest numerical, but non-significant, trend toward an increase in phosphate recovered (20.89% and 21.48%, respectively, versus 18.28% for the LY3358966 group, Figure 6).
In the second approach, feces and different sections of the rat gastrointestinal tract were collected 4 h after dosing 33 P-phosphate to measure the radioactive phosphate recovered in these sections. Since negligible 33 P-phosphate was recovered in feces over this time frame (see Figure 6 for an example), the feces were not further analyzed. The difference in the

| DISCUSS ION
The impaired ability of the kidney to excrete phosphate in CKD and ESRD has pathological consequences, leading to bone, kidney, and  There is conflicting data in literatures on the importance of sodiumdependent phosphate absorption in rats. 31,32 Unlike mice where the majority of NPT2b mRNA and protein expression is found in the ileum, in rats, the majority is found in the jejunum, with some found in the duodenum. 33,34 The rat NPT2b distribution is consistent with the more rapid absorption of phosphate found in human jejunum, compared to the ileum. 35 A role for PiT1, PiT2, and/or another yet to be defined transporter has been proposed for rat intestinal phosphate absorption. 22,23,36,37 In rats, PiT1 mRNA is found in all three segments of the small intestine, but protein is mostly found in the jejunum with some in the duodenum. Low levels of PiT2 mRNA are expressed in all three segments of the small intestine.
PiT2 protein is also found in all three segments. 33,36 In mice, both PiT1 and PiT2 mRNA is similarly expressed in the duodenum, jejunum, and

ACK N OWLED G M ENTS
We express gratitude to Eli Lilly colleagues who contributed to the NPT2b project with their excellent intellectual and technical assistances.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data and protocol will be made available upon request.